Oncogenic Forms of NOTCH1 Lacking Either the Primary Binding Site for RBP-Jκ or Nuclear Localization Sequences Retain the Ability to Associate with RBP-Jκ and Activate Transcription

Abstract

Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (⌬E) or consisting only of the intracellular domain (ICN) were found to activate transcription in cultured cells, presumably through RBP-J response elements within DNA. Both truncated forms also bound to the transcription factor RBP-J in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation required the presence of a weak RBP-J-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-J-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-J, was not needed for oncogenesis or for transcriptional activation. While ICN appeared primarily in the nucleus, ⌬E localized to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreased nuclear localization and increased transcriptional activation by membranebound ⌬E. Transcriptional activation by this mutant form of membrane-bound ⌬E was approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-J at promoter sites.NOTCH1 (also referred to as TAN-1), a homolog of Drosophila NOTCH, was first identified at the breakpoint of a recurrent (7;9)(q34;q34.3) chromosomal translocation associated with a subset of human T cell acute lymphoblastic leukemia/ lymphoma (T-ALL) 1 (1-3). RNA transcribed from the normal NOTCH1 gene is found in most cells but is present at highest levels in developing thymus and brain (2, 4). The product of this gene is a 2555 amino acid type I transmembrane receptor protein of about 350 kDa (p350) containing a series of structural motifs originally described in other polypeptides (Fig. 1), including 36 epidermal growth factor-like repeats and three lin-12-like repeats in the extracellular domain and six ankyrinlike repeats in the intracellular domain. All chromosomal breakpoints in human T-ALLs occur within a single intron dividing the coding sequence for epidermal growth factor repeat 34 and result in overexpression of novel mRNAs containing sequences from the 3Ј end of NOTCH1 (5). These transcripts encode a series of truncated NOTCH1 polypeptides of ϳ100 -125 kDa lacking the epidermal growth factor-like and lin-12-like repeats (5). The oncogenicity of truncated NOTCH1 has been confirmed by inserting cDNAs encoding portions of the NOTCH1 gene into murine bone marrow progenitor cells which were then transplanted into syngeneic r...

Involvement of Notch1 in the development of mouse mammary tumors

“…In most biological assays, the presence of this region has been found to be required to elicit a phenotype Wettstein et al, 1997;Milner et al, 1996), although Notch1 mutants deleted of ankyrin-repeats have been reported to exhibit signi®cant residual activity in some functional assays Aster et al, 1997). Our data also suggest that the ankyrin-repeats are necessary, but not su cient, for transformation of HC11 cells.…”

“…In addition, these results indicated that the 283 bp region (nt 5270 ± 5553, aa 1734 ± 1821) adjacent to the internal surface of the plasma membrane (domain I) and containing the CBF1/RBP-Jk primary binding site (Aster et al, 1997;Hsieh et al, 1996) and one nuclear localization signal (NLS) is critical for transformation. Our data showed that the ankyrin repeats are not su cient by themselves for transformation since mutant F (DTM) and G (M188) in which the six ankyrin repeats were intact could not induce growth in agar of HC11 cells.…”

“…The ankyrin-repeats region is known to bind with some e ectors of Notch (Diederich et al, 1994;Jarriault et al, 1995;Guan et al, 1996;Aster et al, 1997;Fortini and Artavanis-Tsakonas, 1994). In most biological assays, the presence of this region has been found to be required to elicit a phenotype Wettstein et al, 1997;Milner et al, 1996), although Notch1 mutants deleted of ankyrin-repeats have been reported to exhibit signi®cant residual activity in some functional assays Aster et al, 1997).…”

“…However, Notch1 mutants deleted of these sequences have also been reported to have functional activity, indicating the presence of CBF1-independent pathways (Shawber et al, 1996;Aster et al, 1997;Kopan et al, 1994). In contrast, transformation of HC11 cells by Notch1 intra appears to require domain I, since mutant G and mutant F, deleted of most of this domain, were inactive.…”

Neoplastic transformation by Notch is independent of transcriptional activation by RBP-J signalling

“…The Notch-IC module is characterized by three domains, which have been conserved throughout evolution: (i) the RAM domain adjacent to the transmembrane domain is the major docking site for the RBP-J protein (Jarriault et al, 1995;Tamura et al, 1995;Aster et al, 1997); (ii) the ankyrin repeat domain (ANK) adjacent to the RAM domain mediates further protein-protein interactions (Artavanis-Tsakonas et al, 1999); (iii) the C-terminal domain carries two characteristic features: a polyglutamine region (OPA) and a proline, glutamic acid, serine and threonine rich region termed PEST. Recently, intrinsic transcriptional activation capacity has been assigned to the Notch-IC fragment carrying the OPA motif (Kurooka et al, 1998).…”